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cDNA cloning & expression - use ORF or full-length cDNA?


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5 replies to this topic

#1 shojjahd

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Posted 29 January 2009 - 04:01 AM

Hi All,

Please can someone help <_<

I want to express a protein in HEK293 cells via transfection in order to do functional studies (not for purification etc) so I dont think I want it tagged etc etc. But do you use the full length cDNA or the ORF clone? Both are available. I have no clue - please help!!

I am just interested in maximal expression of the active protein and will assay the cells directly for function.

Thanks in advance!

#2 WHR

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Posted 11 February 2009 - 07:45 AM

We used to express the ORF only. Including a Kozak sequence in front of the start codon may helps.

#3 scolix

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Posted 11 February 2009 - 10:41 AM

As suggested, use ORF.

Remember transient transfection will give you something like 1000x overexpression of the protein.

#4 shasa753

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Posted 01 March 2009 - 04:51 PM

Hi,
sorry to bump in with not-exactly-related to the original question. But i have a question about transfection of retroviral vector using fugene6. found an exact post with the exact prob but the link was broken so i couldn't follow on that. Here's the situation:

Need to transfect an LZRS-ires-egfp (~13kb) plasmid into 293GP cell;
Did:

1. optimizing transfection using (ugDNA:ulFugene) 2:9, 3:9, 4:9, 5:9
2. optimize again using (ugDNA:ulFugene) 2:3, 2:6, 2:9, 2:12

all of these in 6-well plate with 40-50% cell confluency. The transfection results was very2 low as seen by microscopy while FACS showed it was ~20-30%. A control transfectin using pEGFPc1 (1ug:9ul) have better results (~80% by microscopy, though funny that FACS says it was just ~40%).

prep of dna:fugene mix and transfection were as follows:

- optimem + fugene (to final vol. 100ul) - incubate RT 5 min,
- optimem/fugene mix + DNA - incubate RT 1 hr
- remove overnight medium from plate and dropwise mix onto cell
- incubate at RT 1 hr with tilting of plate every 15 minutes
- top wells with 2ml complete DMEM medium (10% fcs + antibiotic) and incubate 48hrs before microscopy or facs-ing

Have any idea how else should i troubleshoot the transfection? Really appreciate for any feedbacks.

Thanks in advance.

#5 NemomeN

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Posted 01 March 2009 - 07:17 PM

Hi,
sorry to bump in with not-exactly-related to the original question. But i have a question about transfection of retroviral vector using fugene6. found an exact post with the exact prob but the link was broken so i couldn't follow on that. Here's the situation:

Need to transfect an LZRS-ires-egfp (~13kb) plasmid into 293GP cell;
Did:

1. optimizing transfection using (ugDNA:ulFugene) 2:9, 3:9, 4:9, 5:9
2. optimize again using (ugDNA:ulFugene) 2:3, 2:6, 2:9, 2:12

all of these in 6-well plate with 40-50% cell confluency. The transfection results was very2 low as seen by microscopy while FACS showed it was ~20-30%. A control transfectin using pEGFPc1 (1ug:9ul) have better results (~80% by microscopy, though funny that FACS says it was just ~40%).

prep of dna:fugene mix and transfection were as follows:

- optimem + fugene (to final vol. 100ul) - incubate RT 5 min,
- optimem/fugene mix + DNA - incubate RT 1 hr
- remove overnight medium from plate and dropwise mix onto cell
- incubate at RT 1 hr with tilting of plate every 15 minutes
- top wells with 2ml complete DMEM medium (10% fcs + antibiotic) and incubate 48hrs before microscopy or facs-ing

Have any idea how else should i troubleshoot the transfection? Really appreciate for any feedbacks.

Thanks in advance.


Sounds like your DNA is not very clean. Fugene and 293 cells should be quite easy...normally, I use 2ul of fugene per microgram of DNA...and I use PBS. I try to stay away from optimem as it does contain some protein (~15ug/ml). You should see a fine hazy consistency after you add the fugene to the DNA (appears in a couple minutes or so, especially if you are transfecting ug of DNA) ...and do not remove the media before you add the fugene. Just add it to the media, swirl a few times and that's it. You actually need the media to disperse the DNA around or you will not get an even transfection.

good luck

#6 shasa753

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Posted 02 March 2009 - 03:35 PM

Hi,
sorry to bump in with not-exactly-related to the original question. But i have a question about transfection of retroviral vector using fugene6. found an exact post with the exact prob but the link was broken so i couldn't follow on that. Here's the situation:

Need to transfect an LZRS-ires-egfp (~13kb) plasmid into 293GP cell;
Did:

1. optimizing transfection using (ugDNA:ulFugene) 2:9, 3:9, 4:9, 5:9
2. optimize again using (ugDNA:ulFugene) 2:3, 2:6, 2:9, 2:12

all of these in 6-well plate with 40-50% cell confluency. The transfection results was very2 low as seen by microscopy while FACS showed it was ~20-30%. A control transfectin using pEGFPc1 (1ug:9ul) have better results (~80% by microscopy, though funny that FACS says it was just ~40%).

prep of dna:fugene mix and transfection were as follows:

- optimem + fugene (to final vol. 100ul) - incubate RT 5 min,
- optimem/fugene mix + DNA - incubate RT 1 hr
- remove overnight medium from plate and dropwise mix onto cell
- incubate at RT 1 hr with tilting of plate every 15 minutes
- top wells with 2ml complete DMEM medium (10% fcs + antibiotic) and incubate 48hrs before microscopy or facs-ing

Have any idea how else should i troubleshoot the transfection? Really appreciate for any feedbacks.

Thanks in advance.


Sounds like your DNA is not very clean. Fugene and 293 cells should be quite easy...normally, I use 2ul of fugene per microgram of DNA...and I use PBS. I try to stay away from optimem as it does contain some protein (~15ug/ml). You should see a fine hazy consistency after you add the fugene to the DNA (appears in a couple minutes or so, especially if you are transfecting ug of DNA) ...and do not remove the media before you add the fugene. Just add it to the media, swirl a few times and that's it. You actually need the media to disperse the DNA around or you will not get an even transfection.

good luck



i doubt it's the DNA. prepared it used qiagen midiprep and nanodrop 280/260 reading is 1.87. but using PBS in place of optimem is new. I'll try this one today hopefully this'll work.

thank you :)




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